Princeton University
Invention # 07-2348
Intramembrane proteolysis is a widely conserved regulatory mechanism in
species ranging from bacteria to humans. The first description of intramembrane
proteolysis came from the investigation of cholesterol homeostasis, where the ER
membrane-bound transcriptional factor SREBP must be cleaved by an
integral-membrane protease, known as site-2 protease (S2P). This cleavage
results in the release of the N-terminal
domain of SREBP, which contains a DNA-binding
domain and a transactivation domain. The N-terminal domain of SREBP regulates
transcription of a number of genes that collectively control biosynthesis of
cholesterol and fatty acid. Another prominent example of intramembrane
proteolysis is the proteolytic processing of the amyloid precursor protein (APP)
by the intramembrane protease g-secretase, which is central to the development
of Alzheimer¿s disease. The cleavage product of APP, amyloid b-peptide, exhibits pronounced
toxicity to neuronal cells and is thought to contribute to Alzheimer¿s disease.
More recently, study of epidermal growth factor receptor (EGFR) signaling in Drosophila identified rhomboid as an
essential component in the signal-sending cells. Rhomboid, a putative
intramembrane protease, cleaves the ligand Spitz, which is inactive in its
full-length form, thus regulating EGFR signaling spatially and
temporally.
There are four families of integral membrane proteins that are though to
catalyze intramembrane proteolysis: the serine protease rhomboid,
metalloprotease S2P, aspartyl proteases presenilin (catalytic subunit of
g-secretase) and signal-peptide peptidase. The putative catalytic residues are
predicted to be below the membrane surface and within the hydrophobic core of
the proteases. In this case, since scission of peptide bonds requires the
presence of water molecules, how do hydrophilic water molecules enter the active
site? More importantly, if the active site is within the hydrophobic core of the
proteases, how do the substrate proteins gain excess to the catalytic residues?
Furthermore, are there some common principles that govern all four families of
intramembrane proteases? These fundamental questions need to be addressed by a
series of structures of the proteases at different stages of their
action.
Researchers at Princeton University have determined the crystal structure
of the transmembrane core domain of GlpG, a Rhomboid family intramembrane serine
protease from Escherichia coli. The
protein contains six transmembrane helices, with the catalytic Ser201 located at
the amino-terminus of helix a4 approximately 10 Å below the membrane surface.
Access to water molecules is provided by a central cavity that opens to the
extracellular region and converges on Ser201. One of the two GlpG molecules in
the asymmetric unit exhibits an open conformation at the active site, with the
transmembrane helix a5 bent away from the rest of the molecule. Rigorous structural analysis was
completed which indicates that substrate entry to the active site is most likely
gated by the movement of the transmembrane helix a5 (TMH5). This conclusion may
generally apply to all members of the rhomboid family intramembrane proteases as
well as the other three families of intramembrane proteases, in which gating of
substrate entry is proposed to be provided by a transmembrane
helix.
This invention elucidates the method in which the movement of TMH5 of a rhomboid
intramembrane protease or the corresponding transmembrane helix in other
families of intramembrane proteases is restrained by compounds and inhibitors.
The restrained movement of TMH will result in a reduction of the intramembrane
protease activity of intramembrane proteases. Reduced protease activity of
intramembrane proteases provides a therapeutic treatment for diseases in which
intramembrane proteases play a contributing role. Such diseases could include neurological
disorders, cardiovascular diseases, cancer, and
others.
Publications:
Wu, Z., Yan, W., Feng,
L., Oberstein, A., Yan, H.,Baker, R., Gu, L., Jeffrey, P.,Urban, S., Shi, Y.,
Structural Analysis of a Rhomboid Family Intramembrane Protease Reveals a
Gating Mechanisms for Substrate Entry, Nature, Strucutral Biology, Vol. 13,
#12, December 2006.
Baker,R.P>, Young,K.,
Feng,L., Shi,Y., Urban,S., Enzymatic Analysis of a Rhomboid Intramembrane
Protease Implicates Transmembrane Helix 5 as the Lateral Substrate Gate,
PNAS USA, 2007, May 15:104(20) 8257-62.
Princeton is currently
seeking industrial collaboration to commercialize this technology. Patent
protection is pending.
For more information on
Princeton University invention # 07-2348 please
contact:
Laurie Tzodikov
Office of Technology Licensing and Intellectual
Property
Princeton University
4 New South Building
Princeton, NJ 08544-0036
(609) 258-7256
(609) 258-1159 fax
tzodikov@princeton.edu